The proposed investigation is a pilot study to confirm the in vitro malignant transformation of rat bladder epithelial cells by dibutylnitrosoamine derivatives (N-butyl-N-(3-carboxypropyl) nitrosoamine (BCPN) and N-butyl-N-(4-hydroxybutyl) nitrosoamine (BBN) recently reported by Hashimoto and Kitagawa. Tissue cultures of trypsinized bladder epithelium will be exposed to the nitrosamines and urea as well as nitrosamines and other growth promoting urinary substances. Malignant "transformed" variants detected by their loss of contact-inhibited growth will be characterized morphologically by phase, light, scanning and transmission electron microscopy and for tumorigenicity upon inoculation into syngeneic rats. The surface membrane properties of cells transformed in vitro will be compared with those of cells transformed in vivo and of normal epithelial cells, using the criteria of viral susceptibility, lactoperoxidase-labelled proteins and H3-fucose incorporated glycoproteins. The in vitro technique may be useful 1) to identify the urinary growth promoting factor (s), 2) to screen potential bladder carcinogens and 3) to determine the ultimate form of carcinogenic metabolites of the nitrosamines and their interaction with susceptible urothelium.